RESEARCH Open Access

NLRP7 deubiquitination by USP10promotes tumor progression and tumor-associated macrophage polarization incolorectal cancerBing Li1,2†, Zhi-Peng Qi1,2†, Dong-Li He3,4†, Zhang-Han Chen1,2†, Jing-Yi Liu1,2, Meng-Wai Wong3, Jia-Wei Zhang4,En-Pan Xu1,2, Qiang Shi1,2, Shi-Lun Cai1,2, Di Sun1,2, Li-Qing Yao1,2, Ping-Hong Zhou1,2* and Yun-Shi Zhong1,2*

Abstract

Background: NOD-like receptors affect multiple stages of cancer progression in many malignancies. NACHT, LRR,and PYD domain-containing protein 7 (NLRP7) is a member of the NOD-like receptor family, although its role intumorigenesis remains unclear. By analyzing clinical samples, we found that NLRP7 protein levels were upregulatedin colorectal cancer (CRC). We proposed the hypothesis that a high level of NLRP7 in CRC may promote tumorprogression. Here, we further investigated the role of NLRP7 in CRC and the underlying mechanism.

Methods: NLRP7 expression in human CRC and adjacent non-tumorous tissues was examined by quantitative real-timepolymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. The effect of NLRP7 in CRCprogression was investigated in vitro and in vivo. Proteins interacting with NLRP7 were identified by immunoprecipitationand mass spectrometry analysis while immunofluorescence staining revealed the cellular location of the proteins. Cellularubiquitination and protein stability assays were applied to demonstrate the ubiquitination effect on NLRP7. Cloning andmutagenesis were used to identify a lysine acceptor site that mediates NLRP7 ubiquitination. Cytokines/chemokinesaffected by NLRP7 were identified by RNA sequencing, qRT-PCR, and enzyme-linked immunosorbent assay. Macrophagephenotypes were determined using qRT-PCR, flow cytometry, and immunohistochemistry.

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© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

* Correspondence: zhou.pinghong@zs-hospital.sh.cn;zhongyunshi@yahoo.com†Bing Li, Zhi-Peng Qi, Dong-Li He and Zhang-Han Chen contributed equallyto this work and should be considered co-first authors.1Endoscopy Center, Zhongshan Hospital of Fudan University, 180 FenglinRoad, Shanghai 20032, People’s Republic of ChinaFull list of author information is available at the end of the article

Li et al. Journal of Experimental & Clinical Cancer Research (2021) 40:126 https://doi.org/10.1186/s13046-021-01920-y

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Results: NLRP7 protein levels, but not mRNA levels, were upregulated in CRC, and increased NLRP7 protein expressionwas associated with poor survival. NLRP7 promoted tumor cell proliferation and metastasis in vivo and in vitro andinteracted with ubiquitin-specific protease 10, which catalyzed its deubiquitination in CRC cells. NLRP7 stability andprotein levels in CRC cells were modulated by ubiquitination and deubiquitination, and NLRP7 was involved in theubiquitin-specific protease 10 promotion of tumor progression and metastasis in CRC. K379 was an important lysineacceptor site that mediates NLRP7 ubiquitination in CRC cells. In CRC, NLRP7 promoted the polarization of pro-tumor M2-like macrophages by inducing the secretion of C-C motif chemokine ligand 2. Furthermore, NLRP7 promoted NF-κBnuclear translocation and activation of C-C motif chemokine ligand 2 transcription.

Conclusions: We showed that NLRP7 promotes CRC progression and revealed an as-yet-unidentified mechanism bywhich NLRP7 induces the polarization of pro-tumor M2-like macrophages. These results suggest that NLRP7 could serveas a biomarker and novel therapeutic target for the treatment of CRC.

Keywords: Colorectal cancer, NOD-like receptors, NLRP7, Deubiquitination, Tumor-associated macrophages

BackgroundColorectal cancer (CRC) is one of the most lethal carcin-omas, and its incidence is increasing worldwide [1]. Des-pite an increase in the five-year net survival rates overthe past few decades, the mortality rate of CRC remainshigh, which is mainly due to recurrence and distantorgan metastasis [2, 3]. Therefore, exploring the molecu-lar mechanisms that drive tumor initiation and progres-sion in CRC is critical for developing novel preventiveand therapeutic strategies against this disease.NOD-like receptors (NLRs), which were originally stud-

ied as critical regulators of the immune response, serve aspattern recognition receptors that specifically recognizepathogen-associated molecular patterns (PAMPs) [4]. Re-cent evidence supports the concept that innate immunesignaling plays an important role in cancer progression [5,6]. A significant role for members of the NLR family hasbeen revealed as contributing either directly or indirectly toa variety of traits associated with cancer, including inflam-mation, cell death, tumor growth, angiogenesis, invasion,and metastasis [7–10]. In terms of functionality, somemembers can be initiators of the inflammasome pathway,which canonically activates caspase-1, and IL-1β and IL-18thereafter [11, 12]. However, some other NLRs regulate di-verse biological pathways associated with both inflamma-tion and tumorigenesis through a non-inflammasomepathway [13–15]. For example, the NLRP3 inflammasomesuppresses liver metastasis in CRC by IL-18 signaling [16].However, NLRP3 upregulation in CRC cells can alsopromote epithelial-mesenchymal transition in aninflammasome-independent manner [15]. Therefore,in view of the multiple effects of NLR family mem-bers in cancer cells, a comprehensive understanding isneeded of the multiple signaling pathways in whichthey are involved.Among all members of the NLR family, NLRP7 and its

function are poorly understood. Previous studies ofNLRP7 have investigated effects on trophoblast lineage

differentiation and contributions to hydatidiform moledevelopment in abnormal human pregnancies [17, 18].In a new area of research, a correlation between theNLRP7 gene and ulcerative colitis (UC) was recentlyreported, and NLRP7 expression was found to be upreg-ulated in the intestinal mucosa of patients with inflam-matory bowel disease (IBD) [19, 20]. Considering thatIBD patients have an increased risk of CRC and thefunction of NLRP7 in cancers remains unclear [21], wedecided to explore the contribution of NLRP7 to colo-rectal tumorigenesis.In this study, we found that NLRP7 protein levels, but

not mRNA levels, were upregulated in CRC by analyzingclinical samples. We then proposed the hypothesis that ahigh level of NLRP7 in CRC may promote tumor pro-gression, requiring further investigation. The function ofNLRP7 in promoting tumor cell proliferation and metas-tasis was confirmed. Furthermore, we found that NLRP7in CRC cells interacted with ubiquitin-specific protease10 (USP10), which mediated its deubiquitination,thereby increasing its protein stability. In addition,NLRP7 promoted the polarization of pro-tumor M2-likemacrophages in CRC by inducing the secretion of C-Cmotif chemokine ligand 2 (CCL2) through the activationof the NF-κB signaling pathway. Thus, we demonstratedthat NLRP7 can promote both tumor progression andtumor-associated macrophage polarization in colorectalcancer.

MethodsCell culture and reagentsHCT116 and HT29 cells were cultured in McCoy’s 5Amedium (GibCo) supplemented with 10% FBS. DLD-1and THP-1 cells were grown in RPMI 1640 (GibCo)with 10% FBS. LoVo, SW480, and SW620 cells were cul-tured in DMEM (GibCo) high-glucose medium with10% FBS. All cell lines were purchased from ChineseAcademy of Sciences Cell Bank (Shanghai, China). THP-

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1 cells were differentiated into macrophages by exposureto 200 ng/ml phorbol-12-myristate-13-acetate (PMA)(Sigma-Aldrich) for 48 h [22].

Human tissue specimens and immunohistochemicalanalysisThirty pairs of fresh primary CRC and correspondingnon-tumorous tissues were collected immediately aftersurgical resection at Zhongshan Hospital, FudanUniversity (Shanghai, China). Another 115 formalin-fixed and paraffin-embedded (FFPE) CRC samples andadjacent non-tumorous tissue samples were alsoobtained from Zhongshan Hospital. Written informedconsent was obtained from each patient, and the in-vestigation was approved by the Ethics Committee ofZhongshan Hospital. Tumor-node-metastasis (TNM)staging was performed according to American JointCommittee on Cancer (AJCC) standards [23].Consecutive sections of FFPE tumors were subjected

to immunohistochemistry (IHC) staining using rabbitpolyclonal anti-NLRP7 (Abcam, 1:50) and anti-USP10(Abcam, 1:50) antibodies. A DAB substrate kit was usedaccording to the manufacturer’s instructions. The resultswere scored by two pathologists with no prior know-ledge of patient characteristics. Corresponding to thepercentage of immune-reactive tumor cells (0, 1–5, 6–25, 26–75, and 76–100%, respectively), the staining ex-tent score was on a scale of 0–4. The staining intensitywas scored as negative (score = 0), weak (score = 1),medium (score = 2), or strong (score = 3). By multiplyingthe staining extent score by the intensity score, a scoreranging from 0 to 12 was calculated.

Lentiviral-mediated NLRP7 overexpression andknockdownA lentiviral construct carrying NLRP7 (GeneChem,China) was packaged in 293FT cells. Virus-containingsupernatants were collected and stably transfectedinto SW480 and HT29 cells selected by puromycin(Sigma-Aldrich). Empty vector transfected cells wereused as controls. Two lentiviral constructs containingshort hairpin RNAs (shRNA) were purchased fromGeneChem to specifically establish NLRP7 knockdowncell lines. The shNLRP7 construct or the negativecontrol plasmid was transfected into the 293FT cellline, and virus-containing supernatants were collectedfor subsequent transduction into HCT116 cells andDLD-1 cells. Puromycin (Sigma-Aldrich) was used toselect for stably transduced cells. The sequences ofthe two shRNAs against NLRP7 are as follows: 5′-TTGCTGAAGAGGAAGATGTTA-3′ and 5′-TGGCAAGAAACTGGCAGAAAT-3′.

RNA isolation and quantitative real-time PCR (qRT-PCR)Total RNA was extracted using the TRIzol reagent(Invitrogen), and complementary DNA (cDNA) wassynthesized using a reverse transcription-PCR kit(Roche) according to the manufacturer’s instructions.qRT-PCR was performed with Maxima SYBR GreenqPCR Master Mix (Applied Biosystems) on a Quant-Studio 6 Flex Real-Time PCR System (Applied Biosys-tems). GAPDH was used as an internal control. Theprimers were purchased from Asia-Vector Biotechnol-ogy Company (Shanghai, China), and their sequencesare listed in Additional file 1: Table S1.

RNA sequencingAfter total RNA extraction from DLD-1 and SW480cells, the RNA quality and quantity were assessed byNano Drop and Agilent 2100 bioanalyzer (ThermoFisher Scientific). Strand-specific library constructionand sequencing of ~ 100M paired end.100-bp-long reads were performed at the BGI (Huada

Genomics Institute Co. Ltd., Guangzhou, China). Differ-ential expression analysis was performed using DESeq2software [24]. GO and KEGG enrichment analysis of an-notated differently expressed genes was performed byphyper based on hypergeometric test.

Western blot (WB) analysisQuantified protein lysates were separated by sodium do-decyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoridemembranes. Subsequently, the proteins were blocked with5% bovine serum albumin for 1 h at room temperatureand incubated in primary antibodies at 4 °C overnight.After washing with TBS-T, the membranes were incu-bated in horseradish peroxidase-conjugated secondaryantibody for 2 h. GAPDH and β-actin were used as load-ing controls. The antibodies used are listed in Additionalfile 1: Table S2, and unprocessed original scans of blotsare provided in Additional file 2.

Immunoprecipitation (IP) assayAs described elsewhere [25], whole-cell extracts wereprepared in modified buffer (50 mM Tris-HCl pH 7.5,150 mM NaCl, 5 mM EDTA, and protease inhibitorcocktail) and incubated with the indicated antibodies orIgG at 4 °C for 2 h, and then mixed with 100 μl proteinA/G agarose (Santa Cruz) overnight at 4 °C. Beads werewashed with PBS three times, and bound protein wasdenatured with 1× SDS sample buffer. Then, proteinswere collected and analyzed by SDS-PAGE and westernblotting.

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Mass spectrometry analysisSimilar to the IP assay, cellular protein extracts fromHT29 cells were incubated with anti-Flag-NLRP7followed by and protein A/G agarose beads. Recoveredproteins associated with Flag-NLRP7 or IgG were re-solved by gel electrophoresis. The bands specifically bindto Flag-NLRP7 were excised, and proteomics screeningwas accomplished by mass spectrometry analysis on aMALDI-TOF-MS instrument (Bruker Daltonics).

Immunofluorescence (IF) stainingImmunofluorescent staining was performed as describedpreviously [26]. Briefly, cells were grown on glass-bottom culture dishes until the experimental endpoint.Then, the cells were fixed with 4% paraformaldehyde,then washed three times with PBS, and probed with pri-mary antibody against NLRP7, USP10, or NF-κB p65 at4 °C overnight. After washing with PBS, the cells wereincubated with fluorescence-conjugated secondary anti-body (Invitrogen) for 30 min. Next, the cells werewashed twice with blocking solution, and once with PBS.Anti-fade DAPI solution (Life Technology) was thenadded for DNA staining. The cells were imaged using aconfocal laser-scanning microscope. Images were ana-lyzed for co-localization using CoLocalizer Pro Version3.0.2 according to published protocols.

Cloning and mutagenesisFull-length human NLRP7 cDNA and truncated NLRP7were amplified from a human mRNA pool generated byRT-PCR. Site-directed mutagenesis (KKK275RRR,KRK288RRR, K374R, K379R, K461R, K484R, K502R,and K532R) was performed using the Site-Directed Mu-tagenesis kit (Agilent) according to the manufacturer’sprotocol. The sequences of all primers are available inAdditional file 1: Table S3. After cloning, cDNA wasligated into the hU6-MCS-CBh-gcGFP-IRES-puromycinvector purchased from GeneChem. The sequences wereconfirmed by direct sequencing.

Flow cytometryCells were collected and incubated at 4 °C withflorescence-conjugated antibodies, and fixed and perme-abilized with a fixation/permeabilization solution kit (BDCytofix/Cytoperm) and 1% paraformaldehyde (PFA).Flow cytometry results were analyzed with FlowJo soft-ware. Antibodies used are listed in Additional file 1:Table S2.

Protein stability assayIn different cell treatment groups, cycloheximide wasadded into culture medium and cell lysis was collectedat 0, 2, 4, 8, 12, 16, or 24 h after the treatment of

cycloheximide. Then, the protein was visualized usingimmunoblotting assays.

Cellular ubiquitination assayAs described previously, the cellular ubiquitination as-says were conducted [27]. Briefly, IP assay was used andthe immunoprecipitates were obtained by incubating celllysates with anti-FLAG-NLRP7 and A/G agarose beads.The ubiquitinated FLAG-NLRP7 was visualized usingimmunoblotting assays with anti-HA.

Enzyme linked immunosorbent assay (ELISA)The supernatant from CRC cells and serum from an ani-mal model of metastasis was collected to detect the se-cretion level of CCL2 using an ELISA kit (R&DSystems). ELISA was performed according to the manu-facturer’s instructions, and all experiments were per-formed in triplicate.

Animal studiesThe animal experiment was performed with protocolsapproved by the Institutional Animal Care and UseCommittee of Zhongshan Hospital of Fudan University.For in vivo tumorigenic experiments, HCT116 cells (1 ×106) expressing sh-Ctrl, sh-NLRP7, sh-USP10, or sh-USP10/NLRP7 were injected into the right dorsal flankof five-week-old Balb/c nude mice. Tumor formation innude mice was monitored over a six-week period. Thetumor volume was calculated with the following for-mula: tumor volume = 0.5 × length × width2. For in vivometastasis assays, a small left abdominal flank incisionwas made, the spleen was exteriorized, and the preparedcells (2 × 106 cells) were injected into the spleen with a30-Gauge needle. Then, the injected spleen was returnedto the abdomen and the wound was sutured with 6–0black silk. Six weeks later, all mice were sacrificed andnecropsied for observation of visible metastatic lesionsin the liver. In the metastasis animal assay, the periph-eral blood of mice was collected at necropsy and storedat − 80 °C until analysis. All primary and metastatictumor tissues were harvested for further analysis by IHCstaining.

Statistical analysisDifferences between groups were calculated using Stu-dent’s t-test, the chi-square test, or the Fisher exact test.The probability of differences in overall survival (OS)was ascertained by the Kaplan–Meier method with alog-rank test for significance. Analyses were performedwith SPSS version 22.0 and Graphpad Prism 7.0 statis-tical analysis software. Representative data are shown asthe mean ± SD. P < 0.05 was considered statisticallysignificant.

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Fig. 1 (See legend on next page.)

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ResultsNLRP7 protein with high expression level in CRCpromotes tumor cell proliferation and metastasisNLRP7 mRNA levels did not differ significantly between30 CRC tissues and normal colorectal mucosa by qRT-PCR (Fig. 1a), and was confirmed by analysis of gene ex-pression from the GEO dataset (GSE23878, GSE32323,GSE110223, GSE156355) (Additional file 3: Fig. S1A).However, the protein levels of NLRP7 were significantlyhigher in human CRC tissues than in paired normal tis-sues as determined by western blotting (Fig. 1b) andconfirmed by IHC staining in samples from 115 CRC pa-tients compared with the corresponding adjacent normaltissues (ANTs) (Fig. 1c). The protein expression ofNLRP7 in the CRC cohort was significantly correlatedwith distant metastasis (P = 0.004) and advanced clinicalstage (P = 0.033) (Additional file 1: Table S4). Moreover,Kaplan–Meier survival curve analysis demonstrated thatCRC patients with increased NLRP7 protein expressionhad poorer overall five-year survival (Fig. 1d).Next, we investigated the biological role of NLRP7 in

CRC using cell functional assays. Knockdown of NLRP7significantly decreased DLD-1 cell proliferation com-pared with that in the negative control, whereas NLRP7overexpression significantly increased SW480 cell prolif-eration (Fig. 1e and f, Additional file 3: Fig. S1B). Inaddition, the proportion of apoptotic cells decreased inSW480 cells overexpressing NLRP7 and increased inDLD-1 cells after knockdown of NLRP7 (Fig. 1g,Additional file 3: Fig. S1C). To determine the role ofNLRP7 in CRC metastasis, we performed migration andinvasion assays in vitro. The results showed that overex-pression of NLRP7 promoted the migration and invasionof SW480 and HT29 cells, whereas NLRP7 knockdownin DLD-1 and HCT116 cells had the opposite effects(Fig. 1h and i). Collectively, these results indicated thatupregulated NLRP7 protein in CRC promotes tumor cellproliferation and metastasis.

USP10 interacts with NLRP7 and is highly expressed inCRCTo identify candidate proteins that interact with NLRP7,a protein pull-down assay was performed. One of themost abundant and specific bands was separated andidentified as ubiquitin-specific peptidase 10 (USP10) by

liquid chromatography-mass spectrometry (LC/MS-MS)analysis (Fig. 2a), and all proteins that may physicallyinteract with NLRP7 are listed in Additional file 4. IPassays confirmed that USP10 existed in complexes pre-cipitated with antibody against Flag-NLRP7 comparedwith control IgG (Fig. 2b, upper). Binding of endogenousUSP10 to NLRP7 was validated by IP assay with anti-body against USP10 (Fig. 2b, lower). IF staining showedthat NLRP7 extensively colocalized with USP10 in thecytoplasm (Fig. 2c).To evaluate the expression profile of USP10 in CRC,

we analyzed the GEO database (GSE18105, GSE22598,GSE100179, GSE106582), and the results showed thatUSP10 mRNA was significantly upregulated in CRC(Fig. 2d). USP10 protein levels were increased in repre-sentative CRC patient tissues compared with normal tis-sues (Fig. 2e). To investigate the function of USP10 inCRC, we performed IHC staining for USP10 using ar-chived CRC tissues as described previously. The resultsshowed that the expression of USP10 was elevated andsignificantly positively correlated with NLRP7 expressionin CRC tissues (Fig. 2f-g).

Degradation of NLRP7 is repressed by USP10deubiquitinationTo confirm the positive role of USP10 in the regulationof NLRP7, we investigated the effect of changes inUSP10 expression on NLRP7 in HCT116 cells. USP10upregulation increased NLRP7 protein levels (Fig. 3a).However, USP10 had no significant effect on the mRNAlevels of NLRP7 in CRC cells (Fig. 3b). Considering thatUSP10 is a deubiquitinase that removes ubiquitin fromits substrates [28], we analyzed the effects of USP10 onthe deubiquitination and degradation of NLRP7. Con-sistently, knockdown of USP10 in HCT116 cells signifi-cantly shortened the half-life of the NLRP7 protein inthe presence of the protein synthesis inhibitor cyclohexi-mide (CHX) compared with that under control condi-tions (Fig. 3c). These results suggest that NLRP7 levelsare modulated post-translationally through its degrad-ation. Co-IP experiments showed that USP10 knock-down increased the ubiquitination of NLRP7 in HCT116cells (Fig. 3d).Next, we identified the lysine on NLRP7 that binds to

the Ub moiety in CRC cells. Human NLRP7 truncation

(See figure on previous page.)Fig. 1 NLRP7 protein with high expression level in CRC promotes tumor cell proliferation and metastasis. a NLRP7 mRNA levels in CRC tissuesand the normal colorectal mucosa measured by qRT-PCR. b The protein levels of NLRP7 in CRC tissues and paired normal tissues measured byWB. N: non-tumor, T: tumor. c NLRP7 protein in CRC samples and corresponding adjacent normal tissues confirmed by IHC staining. Scale bars,100 μm. d Kaplan–Meier survival analysis of patients with CRC according to NLRP7 expression (low: score < 6, high: score≥ 6). Knockdown andoverexpression of NLRP7 affected CRC cells proliferation as determined by (e) CCK8 assay, (f) colony formation ability and (g) cell apoptosis. Theresults of transwell assay of the (h) migration and (i) invasion for NLRP7 knockdown and overexpression CRC cells. Scale bars, 100 μm. ***P <0.001, **P < 0.01, and *P < 0.05. NS: no significance

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mutants were cloned into the hU6-MCS-CBh-gcGFP-IRES-puromycin vector, and the protein stability ofectopically expressed mutants was assessed in the pres-ence of the protein synthesis inhibitor CHX. A signifi-cant increase in protein stability was only observed inthe 500C NLRP7 mutant (Fig. 3e-g), suggesting that theNLRP7 ubiquitin acceptor site resides within 10 lysine

residues between aa 250 and 500. Point mutations werethen generated by substituting arginine for lysine at eachcandidate acceptor site. The protein stability of theK379R mutant increased significantly in cells treatedwith CHX (Fig. 3h-i). Co-IP studies confirmed that ubi-quitination was decreased in K379R NLRP7 comparedwith that in wild-type NLRP7 (Fig. 3j). These results

Fig. 2 USP10 interacts with NLRP7 and is highly expressed in CRC. a Protein pulldown experiment for the identification of proteins associatedwith NLRP7 by incubation of Flag-NLRP7-antibody with protein extracts from CRC cells. The red box indicates one of the most abundant bandsas compared with IgG. b IP assay showed that Flag-NLRP7-antibody could pull down USP10 (upper). Correspondingly, USP10-antibody also pulleddown NLRP7 (lower). c Immunofluorescence showed colocalization of NLRP7 (red) and USP10 (green). Nuclei were stained with DAPI (blue). Scalebars, 5 μm. d Analysis of USP10 gene expression in CRC from the GEO dataset. e The protein levels of USP10 in CRC tissues and paired normaltissues measured by WB. N: non-tumor, T: tumor. f The USP10 protein in CRC samples and corresponding adjacent normal tissues confirmed byIHC staining. Scale bars, 100 μm. g Correlation analysis of NLRP7 and USP10 protein expression in CRC tissues. ***P < 0.001

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Fig. 3 Degradation of NLRP7 is repressed by USP10 deubiquitination. a USP10 upregulation increased NLRP7 protein levels, but (b) had nosignificant effect on mRNA levels of NLRP7 in CRC cells. c In cycloheximide (CHX) chase, NLRP7 protein stability was decreased in HCT116 cellswith knockdown of USP10 compared to control. d USP10 knockdown increased the ubiquitination of NLRP7 in HCT116 cells. e Mapping ofNLRP7 using truncation mutants. f Half-life analysis of transfected wild-type and truncation mutant NLRP7-FLAG and (h) transfected K-R pointmutant NLRP7-FLAG in CRC cells. Densitometric analysis of (g) NLRP7 truncation mutant and (i) NLRP7 K-R point mutant signal versus time. jImmunopurified NLRP7-FLAG K379 mutant showed decreased ubiquitination compared to wild-type in HCT116 cells with shUSP10. **P < 0.01,and *P < 0.05. NS: no significance

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Fig. 4 (See legend on next page.)

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suggest that K379 is an important lysine acceptor site in-volved in modulating NLRP7 half-life in CRC cells.

USP10 accelerates the CRC malignant process byupregulating NLRP7To explore the role of NLRP7 in the tumor-promotingfunction of USP10, the effect of NLRP7 overexpression onthe tumorigenic potential of USP10-shRNA lentivirus-stable–transfected HCT116 and SW480 cells was exam-ined. Downregulation of USP10 expression significantlysuppressed the proliferative, migration, and invasive cap-abilities of HCT116 and SW480 cells, whereas overexpres-sion of NLRP7 attenuated the inhibitory effect of USP10silencing (Fig. 4a-c).Tumor xenograft experiments showed that NLRP7 or

USP10 knockdown inhibited the growth of HCT116 tu-mors in vivo, whereas overexpression of NLRP7 inUSP10 knockdown cells had a synergistic effect ontumor growth (Fig. 4d). IHC staining for Ki-67, E-cadherin, and vimentin in xenograft tumors was per-formed and signals were captured under a microscope(Fig. 4e). NLRP7 or USP10 knockdown dramaticallyinhibited liver metastasis in a mouse model. However,NLRP7 overexpression reversed the inhibition of livermetastasis by USP10 knockdown (Fig. 4f-g). Collectively,the in vitro and in vivo results suggest that NLRP7 is in-volved in the effect of USP10 on promoting tumor pro-gression and metastasis in CRC.

NLRP7 promotes CCL2 expression and TAM polarizationTo elucidate the molecular mechanism by which NLRP7promotes CRC progression, CRC cells with stableNLRP7 overexpression and knockdown were subjectedto RNA sequencing (RNA-seq). RNA-seq identified 3065transcripts that were significantly upregulated by NLRP7overexpression (fold change > 2) and 2389 transcriptsthat were significantly downregulated (fold change < 0.5)by NLRP7 knockdown (Fig. 5a). The results showed that398 genes were positively correlated with NLRP7 expres-sion and overlapped between the two groups. The 398transcripts are listed in Additional file 5, and the GOand KEGG enrichment analysis results are shown inAdditional file 3: Fig. S2A and S2B. Cytokines and che-mokines, as downstream signal transduction molecules,are essential for NLR family members to ensure their

function and influence disease outcomes [6, 10, 29, 30].Therefore, we decided to focus on cytokines and chemo-kines that are regulated by NLRP7 in cancer cells.Among 398 transcripts, four cytokines/chemokines, in-cluding CCL2, TNFSF13, CXCL8, and TNFSF15, werepositively correlated with the expression of NLRP7.Among them, CCL2 mRNA detected by qRT-PCR wasdramatically increased in NLRP7-overexpressing cellsand decreased in NLRP7 knockdown cells (Fig. 5b).ELISA with a specific anti-CCL2 antibody confirmedthat CCL2 secretion was induced by NLRP7 overexpres-sion and significantly repressed by NLRP7 knockdown(Fig. 5c). Because high levels of CCL2 are correlated withincreased numbers of TAMs in tumor tissues in manyhuman tumors [31], we speculated that the recruitmentof TAMs by CCL2 is involved in the function of NLRP7in promoting CRC progression.To test the hypothesis that NLRP7 regulates the re-

lease of chemokines by CRC cells to modulate TAMpolarization, THP-1 cells were incubated with condi-tioned medium (CM) collected from different CRC celllines, and the expression of TAM polarization markerswas analyzed. The culture medium from NLRP7 overex-pressing cells promoted the chemotaxis of THP-1 hu-man monocytes (Additional file 3: Fig. S3A). Moreover,macrophages (F4/80) were reduced in the tumor micro-environment (TME) of xenograft tumor tissues inNLRP7 knockdown group compared with control, asdetected by immunohistostaining (Fig. 5d). The CMfrom NLRP7 overexpressing CRC cells, but not the con-trol medium, upregulated the mRNA expression ofVEGF and ARG1 which were used as indicators for thefunctional polarization of TAMs promoted by tumor sig-nals [32] (Fig. 5e). Flow cytometry showed that theCD68(+)/CD206(+) subpopulations were more abun-dant, whereas CD68(+)/CD86(+) subpopulations wereless abundant in cells overexpressing NLRP7 than in thecontrols (Fig. 5f). The opposite effect was observed inTHP-1 cells incubated with CM from NLRP7 knock-down cells.

CCL2 secretion from CRC cells is necessary for NLRP7inducing TAM polarizationTo investigate whether the effect of CRC cells on TAMpolarization is mediated by the secretion of CCL2,

(See figure on previous page.)Fig. 4 USP10 accelerates the CRC malignant process by upregulating NLRP7. a-c NLRP7 overexpression rescued impaired proliferation, colonyformation, migration, and invasive capability caused by USP10 knockdown in HCT116 and SW480 cells. Scale bars, 100 μm. d NLRP7 or USP10knockdown inhibited the growth of HCT116 tumors in vivo, whereas overexpression of NLRP7 in USP10 knockdown cells had a synergistic effecton tumor growth. e Representative IHC images of Ki-67, E-cadherin, and vimentin in different xenograft sections (magnification: × 600). Scale bars,50 μm. f Visible metastatic lesions in the liver, and NLRP7 overexpression reversed the inhibition of liver metastasis by USP10 knockdown. gRepresentative images of hematoxylin-eosin staining sections from metastatic nodules in the liver (magnification: × 200). Scale bars, 25 μm.***P < 0.001, **P < 0.01, and *P < 0.05

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Fig. 5 NLRP7 promotes CCL2 expression and TAM polarization. a Number of altered transcripts detected in CRC cells with either NLRP7overexpression or knockdown. b qRT-PCR verification of transcription of four cytokines/chemokines, including CCL2, TNFSF13, CXCL8, and TNFSF15, in CRC cells with NLRP7 overexpression or knockdown. c ELISA confirmed CCL2 secretion levels in CRC cells with NLRP7 overexpression orknockdown. d Macrophages detected by immunohistostaining of the xenograft tumor tissues from NLRP7 knockdown and control groups(magnification: × 600). Scale bars, 50 μm. e Expression levels of M2-like macrophage marker changes after treatment with culture medium fromNLRP7 overexpressing or knockdown CRC cells. f Flow cytometry was used to evaluate CD68(+)/CD206(+) and CD68(+)/CD86(+) subpopulations.***P < 0.001, **P < 0.01, and *P < 0.05. NS: no significance

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Fig. 6 (See legend on next page.)

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recombinant CCL2 and a CCL2-neutralizing antibodywere added to the CM from NLRP7 knockdown cellsand the CM from NLRP7 overexpressing cells, respect-ively. The results of qPCR and flow cytometry confirmedthat CCL2 induced M2-like macrophage polarization ina dose-dependent manner (Fig. 6a-b). Moreover, the CMfrom SW480 cells treated with CCL2-neutralizing anti-body significantly inhibited the chemotaxis of THP-1cells, which was restored by exogenous CCL2 addition(Additional file 3: Fig. S3B).In animal studies, macrophages were reduced in the

TME of xenograft tumor tissues in NLRP7 and USP10knockdown groups and were increased in the NLRP7overexpressing group (Fig. 6c). The Vegf and Arg1mRNA expression in tissues was higher in NLRP7-overexpressing tumors and lower in sh-NLRP7 and sh-USP10 tumors than in the control group (Fig. 6d). Fur-thermore, IHC staining for the M1 marker CD86showed that accumulation of CD86-positive M1 macro-phages was greater in the stroma of sh-NLRP7 tumorsand sh-USP10 tumors than in that of scrambled shRNAtreated tumors, and NLRP7 overexpression suppressedthe accumulation of CD86-positive M1 macrophages.CD163-positive M2 macrophages were more abundantin NLRP7-overexpressing tumors and less abundant insh-NLRP7 tumors and sh-USP10 tumors than in thecontrol tumors (Fig. 6e). The expression levels of CCL2in peripheral blood and tumor tissues were significantlylower in sh-NLRP7-treated and sh-USP10-treated micethan in control mice and higher in NLRP7-treated micewith USP10 knockdown (Fig. 6f). Thus, data from cellsand animal models suggested that NLRP7 enhancesTAM polarization by promoting CCL2 secretion fromCRC cells.

NLRP7 facilitates transactivation of CCL2 via NF-κBConsidering that CCL2 is a classical NF-κB target gene[25], we next examined whether the effect of NLRP7 onCCL2 expression and monocyte chemotaxis is mediatedby enhanced NF-κB signaling. The NF-κB circuit in-volves p65 phosphorylation and nuclear translocation,which lead to gene activation. Phosphorylation of p65was increased in NLRP7-overexpressing cells, whereasthe protein levels did not change (Fig. 7a). IF analysisdemonstrated that NLRP7 overexpression increased p65

NF-κB accumulation in the nucleus compared with thatin control cells (Fig. 7b). These results suggest thatNLRP7 promotes p65 NF-κB accumulation in the nu-cleus by enhancing its phosphorylation.Next, we determined the effect of NLRP7 on CCL2 ex-

pression in response to NF-κB activation. We confirmedthat the NLRP7-induced expression of CCL2 was signifi-cantly suppressed following NF-κB inhibition using theNF-κB-specific inhibitor CAPE (Fig. 7c). Luciferase re-porter gene analysis showed that ectopic expression ofNLRP7 induced, whereas CAPE suppressed CCL2 tran-scription (Fig. 7d). These results suggest that NLRP7regulates the expression of CCL2 by modulating the NF-κB signaling pathway.Collectively, the present results indicated that NLRP7

in CRC cells interacts with and is deubiquitinated byUSP10, resulting in increased NLRP7 protein stability(Fig. 7e). NLRP7 promoted the secretion of CCL2 by ac-tivating the NF-κB signaling pathway. CCL2 secreted byCRC cells promoted the polarization of pro-tumor M2-like macrophages. Therefore, upregulation of NLRP7 inCRC facilitated tumor cell proliferation and metastasis.

DiscussionNLRs affect multiple stages of cancer progression inseveral malignancies, including colorectal cancer, gastriccancer, hepatocellular carcinoma, and other cancers [6].The exact roles of NLR family members in CRC are di-verse and complex. In this study, we showed that NLRP7is upregulated at the protein level in CRC samples com-pared with matched non-tumor controls, and that over-expression of NLRP7 is associated with poor prognosisof CRC patients. Functional studies in vitro and in vivorevealed that NLRP7 promoted tumor cell proliferationand metastasis. Previous studies focused on the role ofNLRP7 during embryonic development [33]. The presentresults thus expand our knowledge of the role of thisprotein in cancer.At the beginning of this study, we observed an

increase in NLRP7 protein levels without changes inmRNA levels in CRC tissues compared with levels inthe corresponding ANTs. This phenomenon needed tobe explained mechanistically. Protein ubiquitination isan important post-translational modification that reg-ulates the degradation of proteins, but not alteration

(See figure on previous page.)Fig. 6 CCL2 secretion from CRC cells is necessary for NLRP7 induction of TAM polarization. a-b The results of qPCR and flow cytometryconfirmed that recombinant CCL2 rescued the M2-like macrophage polarization effect from CM of NLRP7 knockdown cells, and CCL2-neutralizingantibody suppressed M2-like macrophage polarization effect from CM of NLRP7 overexpressing cells. c Macrophages in xenograft tumor tissuesfrom control, NLRP7 knockdown, USP10 knockdown, and USP10 knockdown with NLRP7 overexpression (magnification: × 600). Scale bars, 50 μm.d Expression level of TAM functional polarization markers in xenograft tumor tissues. e CD86-positive M1 macrophages and CD163-positive M2macrophages in xenograft tumor tissues from four groups (magnification: × 600). Scale bars, 50 μm. f The expression levels of CCL2 in peripheralblood and tumor tissues of animals from four groups. ***P < 0.001, **P < 0.01, and *P < 0.05. NS: no significance

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of mRNA [34]. A recent study showed that STAM-binding protein (STAMBP), a deubiquitinating en-zyme, modulates NLRP7 protein stability by catalyzing

its deubiquitination to increase NLRP7 abundance[35]. Moreover, some other NLR family members, in-cluding NLRP3 and NOD2, were also demonstrated to

Fig. 7 NLRP7 facilitates transactivation of CCL2 via NF-κB. a Phosphorylation of p65 was increased in NLRP7-overexpressing cells, whereas theprotein levels did not change. b Immunofluorescence analysis demonstrated that NLRP7 overexpression increased p65 NF-κB accumulation in thenucleus. Scale bars, 10 μm. c NF-κB-specific inhibitor CAPE suppressed the NLRP7-induced expression of CCL2. d Luciferase reporter gene analysisshowed that ectopic expression of NLRP7 induced, whereas CAPE suppressed CCL2 transcription. e A schematic diagram displays high level ofNLRP7 protein deubiquitination by USP10 promoting tumor-associated macrophage polarization via NF-κB-CCL2 signaling in colorectal cancer.***P < 0.001, **P < 0.01, and *P < 0.05. NS: no significance

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be affected by de-ubiquitinase enzymes (DUBs) [36–39]. Even without experimental validation, the aboveliterature suggests that NLRP7 regulated by deubiquiti-nation is a well-founded hypothesis. Here, we identifiedUSP10 as a critical regulator of NLRP7 deubiquitina-tion and stabilization in CRC cells. We identified K379as an important lysine acceptor site that mediatesNLRP7 ubiquitination in CRC cells. However, a previ-ous study identified KRK288/290 as the main lysine ac-ceptor sites affecting ubiquitination of NLRP7 [35].This discrepancy may be related to differences in thecells and conditions used for analyzing NLRP7, as wellas different biological functions of NLRP7.Several lines of evidence support the diverse roles of

USP10 in tumorigenesis in different cancer types. For ex-ample, in hepatocellular carcinoma, Zhu et al. reportedthat USP10 promotes proliferation [40], whereas Lu et al.found that USP10 suppresses tumor progression by inhi-biting mTOR activation [41]. In the present study, USP10was overexpressed in CRC tissues, as determined by ourdata and public GEO database information. TargetingUSP10 by shRNA inhibited CRC proliferation and metas-tasis, and this effect was reversed by overexpression ofNLRP7. This result indicates that NLRP7 may be a keyeffector molecule for the cancer-promoting effects ofUSP10 in CRC cells.TAMs constitute a plastic and heterogeneous cell

population of the tumor microenvironment, and theyare closely related to cancer progression and resistanceto therapy [42]. Here, we found that NLRP7 overexpres-sion in CRC cells enhanced TAM polarization, whichmay be one of the mechanisms by which NLRP7 pro-motes CRC progression. Polarization of TAMs is reg-ulated by multiple microenvironmental cytokines,growth factors, epigenetic regulators, and other sig-nals derived from tumor and stromal cells [43]. Weshowed that NLRP7 promoted the transcription ofCCL2, and the secretion of CCL2 was increased inNLRP7 overexpressing CRC cells. Several studies re-ported that CCL2 not only promotes tumor invasionand metastasis, but also acts as a macrophage re-cruiter and M2-stimulating factors [44–46]. Weshowed that M2-like macrophage polarization inducedby overexpression of NLRP7 in CRC cells is mediatedby CCL2, and this effect can be attenuated by aCCL2-neutralizing antibody in vitro. Moreover, theprotein levels of CCL2 in peripheral blood were sig-nificantly decreased in sh-NLRP7-treated mice and in-creased in NLRP7-treated mice, whereas M2-likemacrophages were more abundant in NLRP7-overexpressing tumors and less abundant in sh-NLRP7 tumors. These results indicate that high ex-pression of NLRP7 in CRC promotes macrophagepolarization in a CCL2-dependent manner.

Mechanistically, we demonstrated that the NF-κBpathway is essential for CCL2 production induced byNLRP7 in CRC cells. We confirmed that NLRP7 pro-motes the phosphorylation of p65 NF-κB, leading to in-creased nuclear accumulation of p65 NF-κB. NLRs werepreviously shown to modulate NF-κB signaling by inhib-ition or activation [47]. NLRC1 activates NF-κB signaling[48], whereas NLRP12 negatively regulates NF-κB [13].In this study, the translocation of NF-κB from the cyto-plasm to the nucleus promoted by NLRP7 enhanced thebinding of NF-κB to the CCL2 promoter, therebyincreasing its expression and secretion. Previous studiesshowed that the genes encoding CCL2 are downstreamtargets of the NF-κB transcription factor, which arecrucial for cancer progression and metastasis [25, 49].Therefore, the NF-κB-CCL2 signaling axis plays a criticalrole in the effect of NLRP7 on promoting TAMpolarization and CRC progression.

ConclusionIn summary, we identified a potential role of NLRP7 inthe pathogenesis and metastasis of CRC. NLRP7 proteinlevels were increased by USP10-mediated deubiquitinationin CRC cells, which induced the polarization of pro-tumorM2-like macrophages via NF-κB pathway-mediated CCL2secretion. These findings provide a better understandingof the oncogenic mechanisms of NLRP7, and may advancethe development of therapeutic strategies for CRC.

AbbreviationsCRC: Colorectal cancer; TME: Tumor microenvironment; TAMs: Tumor-associated macrophages; CCL2: C-C motif chemokine 2; NLRs: NOD-likereceptors; PAMPs: Pathogen-associated molecular patterns; NLRP7: NACHT,LRR and PYD domains-containing protein 7; UC: Ulcerative colitis;IBD: Inflammatory bowel disease; OS: Overall survival; USP10: ubiquitin-specific-processing protease 10; ANTs: Adjacent normal tissues;IHC: Immunohistochemistry; IP: Immunoprecipitation;IF: Immunofluorescence; CHX: Cycloheximide; CM: Conditioned medium

Supplementary InformationThe online version contains supplementary material available at https://doi.org/10.1186/s13046-021-01920-y.

Additional file 1: Table S1. Primer sequences for real-time PCR. TableS2. The antibodies used in western blot, flow cytometry, and IHC. TableS3. Primer sequences for truncation mutant and site-directed mutagen-esis. Table S4. Clinicopathological correlation of high NLRP7 expressionin CRC.

Additional file 2: Unprocessed original blots.

Additional file 3: Figure S1. (A) Analysis of NLRP7 gene expressionfrom the GEO dataset. (B) Representative images of colony formationassay. (C) Cell apoptosis in NLRP7 knockdown and overexpression CRCcells manifested by flow cytometry analysis. Figure S2. The GO (A) andKEGG (B) enrichment analysis results of 398 genes positively related withNLRP7 expression. Figure S3. (A) Chemotaxis of THP-1 cells treated withculture medium from cells overexpressing NLRP7. (B) CCL2-neutralizingantibody inhibited THP-1 cell chemotaxis, which was restored by exogen-ous CCL2.

Additional file 4: The list of proteins physically interacted with NLRP7.

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Additional file 5: The list of 398 genes that were positively correlatedwith NLRP7 expression in CRC cells.

AcknowledgementsWe would like to acknowledge the reviewers for their helpful comments onthis paper.

Authors’ contributionsYZ and PZ conceived and designed this study; BL, ZQ, DH, ZC, and JZperformed major experiments; MW, EX, JL, and DS assisted with cellexperiments; QS, LY and SC assisted with animal experiments; BL, ZC and JLanalyzed and interpreted the data; BL, ZQ, DH, and ZC drafted themanuscript; YZ and PZ revised the manuscript. The authors read andapproved the final manuscript.

FundingThis work was supported by grants from the National Key R&D Program ofChina (grant nos. 2018YFC1315000, 2018YFC1315005), the National NaturalScience Foundation of China (grant nos. 81672329, 82002515, 81861168036),the Science and Technology Commission Foundation of ShanghaiMunicipality (grant nos. 19411951600, 19411951601), the Dawn Program ofthe Shanghai Education Commission (grant no. 18SG08), and the ShanghaiSailing Program (grant no. 20YF1407200). The funders had no role in thestudy design, data collection/analysis, decision to publish, or preparation ofthe manuscript.

Availability of data and materialsAll data in our study will be available upon reasonable request.

Declarations

Ethics approval and consent to participateThe present study was approved by the Ethics Committee of ZhongshanHospital, Fudan University, and was performed according to the Declarationof Helsinki. All patients recruited into the study signed written informedconsent forms.

Consent for publicationAll authors read and approved the final manuscript.

Competing interestsThe authors declare that they have no conflicts of interest.

Author details1Endoscopy Center, Zhongshan Hospital of Fudan University, 180 FenglinRoad, Shanghai 20032, People’s Republic of China. 2Endoscopy ResearchInstitute of Fudan University, Shanghai 20032, People’s Republic of China.3Endoscopy Center, Xuhui Hospital, Zhongshan Hospital of Fudan University,Shanghai 20031, People’s Republic of China. 4Department ofGastroenterology, Xuhui Hospital, Zhongshan Hospital of Fudan University,Shanghai 20031, People’s Republic of China.

Received: 20 November 2020 Accepted: 21 March 2021

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